1. Field of the Invention
Squalene (SQE) is a triterpenoid hydrocarbon oil, C30H50, that is widely produced by both plants and animals, and is present in human food. SQU is also widely used in skin cosmetics. In humans, SQE serves a as precursor in the synthesis of cholesterol and all of the steroid hormones (Mayes, 1996; Granner, 1996) (FIG. 1). Both SQE and cholesterol are transported in the blood on very low density lipoproteins (VLDL) and low density lipoproteins (LDL) (Miettinen, 1982; Koivisto and Miettinen, 1988). Squalene and cholesterol are also synthesized in the liver and in the epidermis of the skin where SQE comprises a large amount of the oil secreted by sebaceous glands (Stewart, 1992). Because it is a naturally-occurring biodegradable oil, SQE and its hydrogenated derivative squalane (SQA) have each been proposed for use as the oil component of oil-in-water (o/w) emulsions for new generations of adjuvants for vaccines Minutello et al., 1999).
Immunization against a potential antigen such as SQE presents a particular Catch-22 challenge: first, there have never been any previous antibodies developed that could serve as validated positive controls for anti-SQE antibodies, and second, there is no validated assay available for detecting antibodies to SQE. To overcome this difficult dilemma in the present study, the horns of which are the simultaneous lack of positive antibody controls from immunized animals and lack of a validated assay for antibodies to SQE, our first goal was to inject SQE into mice to try to create antibodies that could potentially be validated as having anti-SQE activity. The second goal, namely the creation of monoclonal antibodies that could serve as positive antibody controls, was considered to be a requirement in the ultimate third goal of development of a valid immunoassay for detection of specific antibodies to SQE.
It has been previously reported that SQE incorporated into non-phospholipid liposomes has an adjuvant effect on the induction of antibodies to a non-phospholipid liposomal protein, but the adjuvant effect was not enhanced further by simultaneous incorporation of lipid A (Gupta et al., 1996). Although incorporation of lipid A without SQE into non-phospholipid liposomes was not tested in the latter study, the potent adjuvant effect of liposomal SQE for liposomal protein was clearly shown. This adjuvant effect of liposomal SQE therefore may also have played a role in our liposomes in the induction of antibodies to SQE.
As with 71% cholesterol in liposomes, the biophysical conformation of 71% SQE in our liposomes is not completely clear. Previous work has suggested that SQE locates itself in the most disordered region of liposomes, predominately in the center area of the liposomal bilayer (Lohner et al., 1993). Because of this it has been proposed that SQE adopts a coil rather than an extended conformation when it is located in the bilayer interior. Although relatively small amounts of SQE have a disruptive effect on the liposomal bilayer and lead to formation of tubules having the HII conformation in liposomes containing phosphatidylethanolamine (Lohner et al., 1993), the HII conformation does not occur in liposomes, such as ours, that lack phosphatidylethanolamine. Nonetheless, the reported ability of SQE to lower the transition temperature of phosphatidylcholine and to cause disruption in the stability of the liposomal bilayer (Lohner et al., 1993), together with the high concentrations of SQE combined with lipid A in the liposomes used in this study, may play a role in the potent ability of these liposomes to induce antibodies to SQE.
From a purely structural standpoint, it may not be initially surprising that antibodies to SQE can be induced in a similar manner to those against cholesterol, in view of the striking apparent structural similarity of SQE and cholesterol (FIG. 1). Balanced against this, however, is the observation that the immunogenic epitope of liposome-associated cholesterol is the polar 3-β-hydroxy group in the A ring (Dijkstra et al, 1996), and the fact that SQE not only lacks any closed ring, but is an exceedingly hydrophobic alkene that completely lacks any polar group.
What, if any, are the potential consequences of induction of antibodies to SQE? A recent publication claims to have detected antibodies to SQE in sick but not in healthy individuals (Asa et al., 2000). However, we believe that such a conclusion may be premature, based on a technical critique of the reported Western blot-type assay that was used (Alving and Grabenstein, 2000). Turning again to cholesterol for comparison, SQE, as a precursor in the synthesis of cholesterol, is found nearly everywhere that cholesterol is found, with the apparent exception that SQE probably does not have a structural role in promoting the stability of membranes. As with cholesterol, SQE circulates in the blood as a constituent of LDL and VLDL (Miettinen, 1982; Koivisto and Miettinen, 1988). Naturally-occurring antibodies to cholesterol have been demonstrated to be present in virtually all human serum samples tested, and they have been proposed to have a vital beneficial role in the normal regulation of LDL and VLDL metabolism (Alving and Wassef, 1999).
2. Description of the Related Art
Antibodies to SQE have had great interest in the popular press. Asa et al., described antibodies to SQE in the serum of sick Gulf War Veterans and purported that these antibodies were responsible for their disease [Asa, et al., “Antibodies to Squalene in Gulf War Syndrome,” Exp. Mol. Path. 68:55 (2000)]. Asa's assay has been criticized for technical reasons, which render the reported results as highly questionable or invalid [Alving, et al., Letter to the Editor. Exp. Mol. Path. 68:196 (2000)]. U.S. Pat. No. 6,214,566 (Asa, et al.) discloses an immunoassay for detecting anti-squalene antibodies.
In order to develop a highly reliable assay for antibodies to SQE, we developed murine monoclonal antibodies to SQE to serve as positive controls [Matyas, et al., “Induction and detection of antibodies to squalene,” J. Immunol. Meth. 245:1 (2000)]. These monoclonal were used to develop an assay for measuring antibodies to squalene in human serum.